This project continues long-term studies to define the mechanism and regulation of vitamin B12 uptake in Escherichia coli. The striking feature of the transport of vitamin B12 and the iron siderophore complexes in Gram-negative bacteria is the use of active transport systems of high affinity and substrate specificity operating across the outer membrane. Acquisition of iron by bacteria in host tissues is an important aspect of their ability to cause disease and could provide a promising site for antimicrobial chemotherapy once the mechanism of transport is understood. The energy-dependent step of transport by the cobalamin transporter BtuB across the outer membrane requires action of the TonB-ExbB-ExbD intermembrane energy-coupling complex. Progress in further understanding of the transmembrane topology and the location of protein segments of BtuB important for substrate recognition, TonB binding, and the transport mechanism requires detailed characterization of structure and function by the proposed combination of biophysical and genetic approaches. The structure of BtuB will be determined by completion of the X-ray crystallographic analysis to identify the location of the transmembrane segments and of the extracellular loops that participate in substrate binding and of the periplasmic domain that may interact with TonB. Substrate-induced changes in the dynamics and local environment of conserved or functionally important portions of BtuB will be identified by structure-informed mutagenesis experiments and EPR spectrometry of spin probes. Sites of interaction of BtuB and TonB, and changes in their contact sites in response to substrate binding or to mutations that affect transport function will be investigated by site-directed disulfide bond crosslinking to define further the genetic and biochemical requirements for this contact. Genes whose expression is modulated by the supply of cobalamins share an unprecedented mode of regulation. This regulatory system appears to act primarily at the level of translation initiation, which in turn affects in a specific but secondary process the level and stability of the transcript RNA. Experiments will investigate the formation and regulatory significance: of changes in RNA secondary structure in the transcript for transcript stability and ribosome binding. Reconstitution of the translational regulatory process in vitro will allow detailed probing of changes in RNA secondary structure and the role of transcript segments in ribosome binding and transcript attenuation. Finally the possibility that cobalamin binding to the RNA is the key regulatory signal will be investigated.